The long-term objective of this project is to enhance the understanding of the earliest events in the response of immune system cells towards the presence of allergens (antigens). The initial effector cells in hypersensitive reactions to allergen invasion are mast cells. The signaling pathways in these cells that lead to degranulation and secretion of hormonal mediators causing inflammation and the symptoms of allergic hypersensitivity are well-understood overall, except for the mechanisms involved in initiating these pathways. It is widely accepted that cross-linking by multivalent antigen of IgE antibodies that are bound to the high affinity IgE receptor Fc5RI is necessary for stimulating degranulation by this receptor. How this cross-linking event is recognized at the inner plasma membrane leaflet, however, is not well understood. Biophysical elucidation of transmembrane signal transduction therefore has the potential to contribute to identifying strategies for therapeutic interference with pathological immune cell signaling. A large literature exists emphasizing the role of intra-membrane compositional heterogeneities in cell signaling. Dynamic compositional lateral heterogeneity is a necessary consequence of the non-ideal mixing properties of biological membranes, however, the functional importance of compositional fluctuations, or domains, is unclear. A major hindrance for progress in this important field of research is the conceptual division between the large experimental areas of lipid model membrane and cellular membrane research. We are using a novel approach to examine plasma membrane heterogeneity, consisting of optical microscopic and spectroscopic characterization of micron-sized plasma membrane vesicles obtained from immune cells. Our preliminary data indicate that the membranes of these vesicles can segregate into laterally coexisting fluid domains. Aim #1 is to characterize, by 1H MAS NMR and fluorescence imaging, the biophysics of fluid domain formation in plasma membranes depending on plasma membrane composition and a variety of additional physiologically relevant control parameters, including receptor crosslinking. This characterization will prepare us to achieve Aim #2 where we will quantitatively examine, by confocal fluorescence microscopy, the molecular details of how signaling proteins distribute among membrane domains. The resulting partition coefficients will allow for critical re-examination of current models for transmembrane signaling transduction, as well as motivate the development of mathematical models for the fine tuning of signaling fidelity by dynamic compositional heterogeneities. In Aim #3, we will compare the conditions governing domain formation in plasma membrane vesicles to cell signaling capacities in live cells and will clarify whether membrane composition poised near a mixing/demixing transition is an important principle in amplifying immune response to stimulation. Allergies affect more than 50 million Americans and cause significant suffering and costs to the health care system[1]. The absence of curing treatments is due in part to the lack of understanding how allergens stimulate immune system responses. Our research has the potential to elucidate mechanistic aspects of the earliest molecular events following allergen invasion and could therefore help identify strategies for preventing and treating allergic hypersensitivity.